Cytomegalovirus (CMV) is a ubiquitous herpesvirus of complex pathogenicity involving acute, chronic, and latent infections. Reactivation of CMV is clinically problematic, especially in bone marrow transplant recipients. Only a few CMV genes which explicitly function to regulate virus-host interactions are identified. Studies with murine CMV (MCMV) affords the opportunity to map and characterize viral genes which regulate pathogenesis in a natural host. Toward this goal, we have identified four open reading frames (ORFs m138-M141) within the MCMV genome which regulate viral expression in macrophages and mice but are nonessential for replication of the virus in fibroblasts. A mutant virus deleted of these ORFs (RV10) replicates poorly in macrophages due to a defect in virus entry and/or immediate early gene expression. In vivo, RV10 fails to grow in the macrophage-rich environment of the spleen and is nonlethal for immunodeficient mice. These findings attest to the pivotal importance of this gene region for viral infectivity of macrophages and target organs in vivo. This application aims to 1) identify among the four MCMV ORFs the gene(s) which confers optimal growth of the virus in macrophages and mice, 2) characterize the function of the gene product(s) in regulating MCMV gene expression in macrophages, and 3) to define the functions of macrophages in regulating early MCMV pathogenesis. Using the new mutant virus with a defined mutation, we will assess the role of the gene of interest in the earliest stages of MCMV infection of macrophages: entry and immediate early gene expression. We will also evaluate the role of tissue macrophages as initial target cells for MCMV infection, and as producers/inducers of early, antiviral cytokines. These studies will apply viral genetics to a novel in vivo model of MCMV pathogenesis to contribute to our understanding of how persistent herpesviruses evolved to control their fate within the infected host.